Bottom Line:
In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures.We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect.This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck).

ABSTRACTLaminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, we observed that while alpha3beta1 are known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation.

Figure 5: Inhibition by soluble laminin 5 (lam5) of early kinases phosphorylation events activated via the CD3–TCR complex. Jurkat cells at 20 × 106/ml in RPMI 5% FCS were incubated with Abs at 37°C for 2.5 min, solubilized, and lysates were immunoprecipitated with (a) a pAb anti–Zap 70, (b) a pAb anti-lck, (c) a pAb anti-fyn. After SDS- PAGE separation and electrotransfer onto Immobilon P membrane, proteins were incubated with HRP conjugated anti-phosphotyrosine Ab (APY) followed by ECL. For densitometric analysis of APY immunoblotting, control values were reduced to 1 in order to compare signals after activation. Values are mentioned under each lane. For reprobing, the membranes were submerged in stripping buffer, blocked and immuno-detected with (a) pAb anti–Zap 70, (b) pAb anti-lck, and (c) pAb anti-fyn was performed. Reactions were revealed by incubating membranes with GAR HRP followed by ECL. Laminin 5 (lam5) was added in soluble form at 1 μg/ml for Zap 70 and fyn analysis and 5 μg/ml for lck analysis.

Mentions:
We tested soluble laminin 5 effects on early kinase phosphorylation events. Since Jurkat cells express a low density of α6β4 we selected by cytofluorometry cells expressing α6β4 in similar density as CD3high thymocytes (not shown). These cells were activated with a CD3 mAb (×3, 10 μg/ml) during 2.5 min at 37°C, with or without soluble laminin 5. Immunoprecipitation of Zap 70, p56lck, or p59fyn was carried out on activated cell lysates, after which precipitated proteins were transferred on Immobilon. Membranes were then incubated with HRP conjugated anti-phosphotyrosine antibody. Results were revealed by ECL system. We observed that the addition of 1 μg/ml of soluble laminin 5 was sufficient to induce an inhibition of Zap 70 (Fig. 5 a) and p59fyn tyrosine phosphorylation (Fig. 5 c). By contrast, the addition of soluble laminin 5, up to 5 μg/ml, a concentration fully efficient in proliferation assays, did not induce a significant inhibition of p56lck phosphorylation (Fig. 5 b). Membranes were stripped, blocked, and reprobed with the precipitating antibody, i.e., respectively anti–Zap 70 (Fig. 5 a), anti-lck (Fig. 5 b), and anti-fyn (Fig. 5 c), respectively, in order to verify amounts of precipitated material in each lane.

Figure 5: Inhibition by soluble laminin 5 (lam5) of early kinases phosphorylation events activated via the CD3–TCR complex. Jurkat cells at 20 × 106/ml in RPMI 5% FCS were incubated with Abs at 37°C for 2.5 min, solubilized, and lysates were immunoprecipitated with (a) a pAb anti–Zap 70, (b) a pAb anti-lck, (c) a pAb anti-fyn. After SDS- PAGE separation and electrotransfer onto Immobilon P membrane, proteins were incubated with HRP conjugated anti-phosphotyrosine Ab (APY) followed by ECL. For densitometric analysis of APY immunoblotting, control values were reduced to 1 in order to compare signals after activation. Values are mentioned under each lane. For reprobing, the membranes were submerged in stripping buffer, blocked and immuno-detected with (a) pAb anti–Zap 70, (b) pAb anti-lck, and (c) pAb anti-fyn was performed. Reactions were revealed by incubating membranes with GAR HRP followed by ECL. Laminin 5 (lam5) was added in soluble form at 1 μg/ml for Zap 70 and fyn analysis and 5 μg/ml for lck analysis.

Mentions:
We tested soluble laminin 5 effects on early kinase phosphorylation events. Since Jurkat cells express a low density of α6β4 we selected by cytofluorometry cells expressing α6β4 in similar density as CD3high thymocytes (not shown). These cells were activated with a CD3 mAb (×3, 10 μg/ml) during 2.5 min at 37°C, with or without soluble laminin 5. Immunoprecipitation of Zap 70, p56lck, or p59fyn was carried out on activated cell lysates, after which precipitated proteins were transferred on Immobilon. Membranes were then incubated with HRP conjugated anti-phosphotyrosine antibody. Results were revealed by ECL system. We observed that the addition of 1 μg/ml of soluble laminin 5 was sufficient to induce an inhibition of Zap 70 (Fig. 5 a) and p59fyn tyrosine phosphorylation (Fig. 5 c). By contrast, the addition of soluble laminin 5, up to 5 μg/ml, a concentration fully efficient in proliferation assays, did not induce a significant inhibition of p56lck phosphorylation (Fig. 5 b). Membranes were stripped, blocked, and reprobed with the precipitating antibody, i.e., respectively anti–Zap 70 (Fig. 5 a), anti-lck (Fig. 5 b), and anti-fyn (Fig. 5 c), respectively, in order to verify amounts of precipitated material in each lane.

Bottom Line:
In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures.We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect.This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck).

ABSTRACTLaminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, we observed that while alpha3beta1 are known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation.